Cyclin-dependent kinase 5 (Cdk5) is widely expressed although kinase activity has been described preferentially in neuronal systems. Cdk5 has an impact on actin polymerization during neuronal migration and neurite outgrowth and deregulation of the kinase has been implicated in the promotion of neurodegeneration. Recently it was shown that Cdk5 modulates dopamine signaling in neurons by regulating DARPP-32 function. In addition, Cdk5 phosphorylates munc-18 and synapsin I, two essential components of the exocytotic machinery. We have shown by reverse transcriptase-polymerase chain reaction, immunocytochemistry, and Western blotting that Cdk5 is present in the insulin-secreting pancreatic ␤-cell. Subcellular fractionation of isolated ␤-cells revealed a glucose-induced translocation of membrane-bound Cdk5 protein to lower density fractions. Inhibition of Cdk5 with roscovitine reduced insulin secretion with ϳ35% compared with control after glucose stimulation and with ϳ65% after depolarization with glucose and KCl. Capacitance measurements performed on single ␤-cells that expressed a dominantnegative Cdk5 mutant showed impaired exocytosis. The effect on exocytosis by Cdk5 appeared to be independent of changes in free cytoplasmic Ca 2؉ concentration. Taken together these results show that Cdk5 is present in ␤-cells and acts as a positive regulator of insulin exocytosis.Insulin is stored in secretory granules in pancreatic ␤-cells and upon stimulation with secretagogues insulin is released by exocytosis. Exocytosis has been suggested to be mediated by the same core fusion machinery that traverses intracellular membrane traffic in all cells (1-3). It was reported that the membrane fusion event required the N-ethylmaleimide sensitive factor (NSF), 1 and soluble NSF Attachment Proteins, ␣-, ␤-, and ␥-SNAP (1, 2). In addition to NSF and SNAPs, a 7 S core complex with SNAp receptors or "SNARE" proteins corresponding to the vesicle component synaptobrevin/vesicular-associated membrane protein, as well as the plasma membrane proteins SNAP-25 (synaptosomal-associated protein of 25 kDa) and syntaxin were necessary for neuronal exocytosis (3). The SNARE hypothesis proposes that the SNARE proteins form trans-complexes between adjacent membranes, thereby forcing them to proximity. After association of ␣-SNAP, the ATPase NSF completes the reaction by disassembling the SNARE complex leading to membrane bilayer mixing (3). More recently, trans-SNARE pairing and NSF activity has been suggested to act either prior to docking of vesicles or after membrane bilayer mixing (4 -9). Initially the SNARE proteins were regarded as neuron-specific. However, syntaxin, SNAP-25, synaptobrevin/ vesicular-associated membrane protein, and other synaptic proteins regulating neuronal exocytosis have also been identified in pancreatic ␤-cells, suggesting that the mechanism for insulin secretion is similar to that of neurotransmitter release from synaptic vesicles in neurons (10 -13). Regulated secretion of insulin from pancreatic ␤-cells has to be tightl...

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine protein kinase that requires association with a regulatory protein, p35 or p39, to form an active enzyme. Munc18-1 plays an essential role in membrane fusion, and its function is regulated by phosphorylation. We report here that both p35 and p39 were expressed in insulin-secreting ␤-cells, where they exhibited individual subcellular distributions and associated with membranous organelles of different densities. Overexpression of Cdk5, p35, or p39 showed that Cdk5 and p39 augmented Ca 2؉ -induced insulin exocytosis. Suppression of p39 and Cdk5, but not of p35, by antisense oligonucleotides selectively inhibited insulin exocytosis. Transient transfection of primary ␤-cells with Munc18-1 templates mutated in potential Cdk5 or PKC phosphorylation sites, in combination with Cdk5 and the different Cdk5 activators, suggested that Cdk5/p39-promoted Ca 2؉ -dependent insulin secretion from primary ␤-cells by phosphorylating Munc18-1 at a biochemical step immediately prior to vesicle fusion.Exocytosis of insulin from pancreatic ␤-cells has been suggested to be mediated by the same core fusion machinery that controls all membrane fusion events in organisms ranging from yeast to human (1-3). The soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) 1 proteins are essential components of this machinery. Proteins localized in the membrane of the transported vesicle (v-SNAREs) specifically interact with proteins in the target membrane (t-SNAREs). In neurotransmitter release from synaptic vesicles, the plasma membrane-associated proteins syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) interact with the vesicular component synaptobrevin/vesicular-associated membrane protein (VAMP) (reviewed in Refs. 4 -6). Several synaptic proteins regulating neuronal exocytosis including syntaxin, SNAP-25, VAMP, and Munc18-1 have also been identified in pancreatic ␤-cells, supporting the idea that the molecular machinery regulating insulin secretion is similar to that of neurotransmitter release from synaptic vesicles (7-11).There are a series of discrete biochemical steps leading to trans-SNARE complex formation and vesicular fusion. The vesicles need to be transported and targeted to the cell surface where they are docked, primed, and finally fused with the plasma membrane. Munc18-1, a member of the sec1/Munc18 protein family has emerged as a critical regulator of exocytosis (12)(13)(14). Indeed, Munc18-1 is shown to be important for vesicle trafficking and essential for synaptic transmission since both synaptic transmission and spontaneous neurotransmitter release is abolished in neocortical neurons from Munc18-1-null mouse mutants (15). Albeit essential for regulated exocytosis, it has been demonstrated both in pancreatic ␤-cells and in neuronal systems, that Munc18-1 may also serve as a negative regulator of secretion (11,16,17). Munc18-1 binds to syntaxin 1 and might thus negatively regulate syntaxin 1 function if the expressi...

Regulatory volume decrease (RVD) in detached cerebellar astrocytes in culture after acute exposure to hyposmolarity was characterized in this and the accompanying paper [H. Pasantes-Morales, R. A. Murray, R. Sanches-Olea, and J. Moran. Am. J. Physiol. 266 (Cell Physiol. 35): C172-C178, 1994]. RVD was independent of extracellular calcium, was accelerated at pH 8-9 and retarded at pH 6, and was reduced at temperatures < 18 degrees C. The cationic pathway activated by hyposmolarity was specific for K+ and Rb+, since RVD was abolished and secondary swelling occurred when these ions replaced Na+. However, Li+, choline, tris(hydroxymethyl)aminomethane, and glucosamine, all as Cl- salts, did not affect RVD. The anion pathway was unselective, since RVD was inhibited when NaCl was replaced by anion K+ salts with a permeability rank of SCN- = I- > NO3- > Cl- > benzoate > acetate >> SO3- > gluconate. RVD was unaffected by bumetanide (50 microM) and weakly inhibited by furosemide (2 mM). Quinidine but not other K+ channel blockers inhibited RVD, and its effect was reversed by gramicidin. RVD was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and dipyridamole but not by diphenylamine-2-carboxylate or anthracene-9-carboxylate. These results suggest that diffusion possibly via channels rather than cotransporters is involved in the swelling-activated K+ and Cl- fluxes. Gramicidin did not change astrocyte volume in isosmotic conditions, but greatly accelerated RVD, suggesting that low Cl- permeability in isosmotic conditions markedly increases by swelling, thus making K+ permeability the rate-limiting step for RVD.

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